The long term objective of the proposed research is to apply the techniques of somatic cell genetics (isolation of variant cell lines, somatic cell hybridization) to a study of the mechanism of action of insulin and insulin-like growth factors. We have shown previously that the mouse melanoma cell line PG19 is unresponsive to the growth-stimulatory action of insulin and MSA (a rat insulin-like growth factor that is closely related to human IGF-II). Insulin and MSA action in the PG19 melanoma cells appears to be blocked or uncoupled at a postreceptor step. The first part of the proposed research will be aimed at identifying the exact biochemical step at which the response to insulin is blocked in the melanoma cells and at which complementation for the growth response occurs in the hybrids. Our major effort in this regard will involve an examination of intracellular mediators of insulin action in the melanoma and hybrid cells. An effort will be made to quantify mediators involved in the stimulation of both phosphorylation and dephosphorylation reactions. A long term purpose of these experiments is to identify intracellular mediators involved in the regulation of cell growth by insulin. The second part of the proposed research will involve mapping of the human genes for the insulin, IGF-I and IGF-II receptors. In this project, we will use monoclonal antibodies directed against the receptors to identify the receptors on the surface of human x mouse somatic cell hybrids. The hybrid clone panel that we are using for this project was developed by crossing human fibroblasts with the mouse A9 cell line. The aim of the third part of the project will be to isolate variants of the H4-II-E-C3' rat hepatoma cell line having a defective growth response to insulin. The major emphasis of this part of the project will be to isolate variants having postreceptor defects in insulin action. The relevance of this project to clinical medicine is that it may in the long term lead to an improved understanding of the biochemical and genetic "pathway" of insulin and IGF action. Among the human diseases for which this improved understanding may ultimately lead to improved treatment are diseases involving insulin resistance, including a common disease (type II diabetes mellitus), and a number of rare syndromes characterized by severe insulin resistance, such as lipoatrophic diabetes, leprechaunism, and type A syndrome of insulin resistance and acanthosis nigricans.